Figure 5.

ARF6 activation and interaction with cytohesin-2 decrease upon platelet stimulation. Washed platelets (4 × 10⁸ mL⁻¹) were stimulated with 0.2 U mL⁻¹ αT and lysed at the indicated time-points and ARF6-GTP was pulled down (PD) using GGA3-GST beads (A). Alternatively, platelets were incubated for 15 min with 0.2% DMSO vehicle (Veh) or 2 μm BIM IX prior to stimulation with 0.2 U mL⁻¹ αT and 10 μm ADP, followed by ARF6-GTP pull down (Bi) or cytohesin-2 co-IP (Ci). Samples were immunoblotted for ARF6 and cytohesin-2 (CYH2). Densitometry was performed using ImageJ software and levels of ARF6-GTP (Bii) and ratios of ARF6 over CYH2 were calculated (Cii). *P < 0.05 (Student's paired t-test), mean ± SEM, n ≥ 4. Shown are representative blots. The arrows (➤) indicate the 47 kDa CYH2 band.